JPH11509409A - Process for the production of riboflavin using microorganisms with altered isocitrate lyase activity - Google Patents

Abstract

  (57) [Summary] By culturing a riboflavin-producing microorganism in a nutrient medium and subsequently isolating the riboflavin produced, the endogenous isocitrate lyase (ICL) activity has been altered to produce riboflavin microbially. A method for microbiologically producing riboflavin, which comprises using a microorganism.

Description

The present invention relates to a method for producing riboflavin using a microorganism having an altered isocitratriase activity. Vitamin B _2, which is referred to as riboflavin, is essential for human beings and animals. In the case of vitamin B ₂ deficiency, inflammation of the oral and pharyngeal mucosa, cracks in the corners of the mouth, irritation of the itch and inflammation of the skin wrinkles, in particular skin damage, conjunctival inflammation, reduced vision and corneal opacity appear. In infants and children, growth arrest and weight loss appear. Therefore, vitamin B _2, especially as vitamin preparations during vitamin deficiency, as well as economically important as feed additive. At the same time, it is used as a food pigment, for example, in mayonnaise, ice cream, pudding and the like. Riboflavin is produced chemically or microbially. Riboflavin is usually obtained in chemical processes as a pure end product in a multi-step process, in which case relatively expensive starting materials, for example D-ribose, must be used. Another method for chemical production of riboflavin is the production of this substance by microorganisms. As starting materials for the microbial synthesis, replenishable raw materials, such as vegetable oils, can be used. The production of riboflavin by fermentation of fungi, for example Ashbya gossypii or Eremothecium ashbyii, is known (The Merck Index, Windholz et al., Eds. Merck & Co., p. 1183, 1983); However, yeasts such as Candida or Saccaromyces and bacteria such as Clostridium are also suitable for riboflavin production. Bacterial strains that overproduce riboflavin are described, for example, in EP 405 370, in which the strain is transformed from Bacillus subtilis into riboflavin-biosynthesis-gene. Obtained by However, this prokaryote-gene is unsuitable for riboflavin recombinant production processes using eukaryotes such as Saccharomyces cerevisiae or Ashbya gossypii. World Intellectual Property Organization (WO) 93/03183 describes the cloning of a gene specific for riboflavin-biosynthesis from the eukaryote Saccharomyces cerevisiae. This gene can be used to construct a recombinant eukaryote that allows for efficient riboflavin production. However, as a rule, starting materials and substrates for riboflavin synthase are present in limited amounts in the microorganism, so that despite an increase in riboflavin biosynthetic activity, no increase in riboflavin production is achieved. It was therefore an object to obtain an improved microbial riboflavin production method using microorganisms that have no or little substrate restriction and thus allow for enhanced riboflavin production. This object is achieved according to the invention by modifying the microorganism used by altering its endogenous isocitrate lyase activity. The change can be ascertained for the unchanged starting strain. There are many possibilities to obtain such microorganisms with altered ICL activity. One possibility is to alter the endogenous ICL gene to encode an enzyme with enhanced ICL activity relative to the starting enzyme. Elevated enzyme activity can be achieved, for example, by effecting enhanced substrate conversion by altering the catalytic center or by abolishing the action of the enzyme inhibitor. Also, increased enzyme activity can be induced, for example, by increasing enzyme synthesis by gene amplification or by eliminating factors that inhibit enzyme biosynthesis. Endogenous ICL activity is advantageously enhanced by mutation of the endogenous ICL gene. Such mutagenesis can be obtained omnidirectionally by typical methods, for example by UV irradiation or mutagen-causing chemicals, or by genetic engineering methods, such as deletions, insertions or substitutions. ICL gene expression is increased by increasing ICL gene copy number and / or by enhancing regulators that positively affect ICL gene expression. Thus, an enhancement of the regulatory elements on the transcription plane can advantageously be performed, wherein strong transcription signals, such as promoters and enhancers, are used. At the same time, however, it is also possible to enhance translation, for example by improving the stability of the m-RNA. To increase the gene copy number, the ICL gene is advantageously incorporated into a gene construct or vector having a regulatory gene sequence arranged in the ICL gene, in particular a regulatory gene sequence that enhances gene expression. Subsequently, the riboflavin-producing microorganism is transformed with a gene construct containing the ICL gene. The ICL gene is advantageously isolated from a microorganism, in particular from Acibia gossypii. However, for the isolation of this gene, all other organisms whose cells have the glyoxylate cycle recruitment sequence and thus contain the isocitrate lyase, as well as plants, are used. Isolation of this gene can also be accomplished by homologous or heterologous complementarity in the ICL gene deficient mutant, or by heterologous probing or PCR using heterologous primers. For subcloning, the insertion of the complementing plasmid can subsequently be minimized by appropriate cleavage with restriction enzymes. After sequencing and identification of the putative gene, appropriate subcloning is performed by fusion PCR. A plasmid having the fragment thus obtained as an insert is introduced into an ICL gene deficient mutant, which is tested for the function of the ICL gene. The functional construct is subsequently used for transformation of the riboflavin producer. After isolation and sequencing, an isocitrate lyase gene having the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 or an allelic variant thereof is obtained. Allelic variants include functional derivatives obtained, in particular, by deletion, insertion or substitution of nucleotides, from the sequence set forth in SEQ ID NO: 1, wherein ICL activity remains obtained. The isocitrate lyase gene has previously been linked to a promoter or a substantially similar DNA sequence, in particular a nucleotide sequence of nucleotides 176 to 550 according to SEQ ID NO: 1. Thus, for example, a promoter which differs from the promoter having the listed nucleotide sequence by one or several nucleotide exchanges by one or several insertions and / or by one or several deletions in the gene, Can be linked in advance without affecting the function or effectiveness of the In addition, the promoter may be made more efficient by altering its sequence, or may be completely replaced by a more effective promoter. The ICL gene can further be arranged with a regulatory gene sequence or a regulatory gene that enhances, in particular, ICL gene activity. That is, the ICL gene can have a so-called "enhancer" that results in enhanced ICL gene expression, for example, via improved interaction between RNA polymerase and DNA. One or several DNA sequences can be ligated before and / or after to an isocitrate lyase gene with or without a preconnected promoter or with or without a regulatory gene. Yes, so this gene is contained in one gene structure. Cloning of the ICL gene results in a plasmid or vector containing the ICL gene and, as described above, suitable for transformation of a riboflavin producer. The cells obtained by transformation (preferably transformed cells of Acibia gossypii) are placed on the chromosome in a form which allows the gene to be replicated, ie in an additional copy, wherein the gene copy is genomic by homologous recombination. And / or contained on a plasmid or vector. Another possibility of producing microorganisms with altered ICL activity consists in producing and selecting microorganisms which are resistant to substances which act in an inhibitory way on ICL. Inhibitors of isocitrate lyase (ICL) are known to those skilled in the art and are listed, for example, in Handbook of Enzyme Inhibitors Publisher: Hellmut Zollner, Verlag Chemie, Weinheim, 1993, p. Particularly preferred inhibitors are phosphoenolpyruvate (PEP), 6-P-gluconate, maleate, especially itaconic acid and oxalate. By the way, when a microbial strain producing riboflavin is cultured in the presence of such an inhibitor, it is surprisingly found that riboflavin formation is inhibited. This is indicated by the formation of colonies that remain white on the culture plate without becoming yellow. Thus, strains that are resistant to isocitrate lyase inhibition can be easily identified in this manner. This is because such strains form riboflavin even in the presence of the inhibitor, thus producing yellow colonies. Such strains can be generated by spontaneous mutations or the corresponding mutations can be induced by conventional methods, for example by chemical or UV irradiation. Thus, a microbial strain that precipitates the increased riboflavin content in the medium can be obtained. Among the resistant strains with enhanced riboflavin formation, particularly in DSM, No. The Acibia gossypii strain deposited at 10067 was obtained. In the process according to the invention, fungi are advantageously used as microorganisms. Suitable fungi (Pilze) are described, for example, in Indian Chem. Engr. Section B. 37, No. 1, 2 (1995), p. 15, table 6. In particular, those belonging to the genus Pichia, Eremothetium and Ashbya, particularly Ashibia gossypii, are preferred. However, microorganisms other than fungi, such as bacteria, especially Indian Chem. Engr. Section B. 37, No. 1, 2 (1995), p. 16, table 6, Table 6 can also be used. Example 1 Preparation of a Genomic Gene Library from Acibia gossypii For the preparation of a genomic DNA library, chromosomal DNA was isolated by the method of Wright and Philip psen (1991, Gene 109: 99-105). The DNA was partially digested with Sau3A and fractionated on a saccharose-density gradient. The largest fragment (FIG. 4) was cut with Bam HI. Coli, S.P. Ligated with the cerevisiae shuttle vector YEp352 (JE Hill et al., 1993, Yeast 2: 163-167). Using this ligation component, E. coli. E. coli DH5a was transformed. From the plate, 3600 colonies that could be identified as clones with the plasmid carrying the insert by their white color were isolated using Ampicillin and X-Gal. In experiments with 30 such randomly selected clones, in fact, all have plasmids, these plasmids have inserts ranging in size from 7 to 18 kb and all inserts are different. This proved to be recognizable by the restricted model. Based on the 7 × 10 ³ kb size of the Acibia gossypii genome, the probability that each gene is contained in this gene library is 97% to 99.99%. Each 100 clones were subjected to large smear culture on agar plates, and then plasmids were prepared as pools. As a result, the gene library consisted of plasmid pool 36. Example 2 Selection of Gene Library Fragments Containing icll Using plasmid preparations of the gene library, the yeast Saccharomyces cerevisiae ICL1d ura3 (fs) (E. Fernández et al., 1992, Eur. Biochem. 204: 983-990). This mutant is split in the ICL1 gene and has an in-frame mutation in the ura3 gene. This genotype indicated that the strain was unable to grow on ethanol as a carbon source and exhibited uracil auxotrophs. In a first step, yeast cells transformed with this gene library were selected on minimal medium with glucose as the sole carbon source. Since this minimal medium did not contain uracil, only cells that had taken up the plasmid could grow based on the ura3 gene present on the plasmid. At this stage, 1900 clones were obtained. This was transferred by replica plating onto a minimal medium containing ethanol as the sole carbon source. Since the isocitrate lyase as a supplementary enzyme is unconditionally required for growth on ethanol, only the clone having the ICL gene was able to grow on the plasmid. Two clones growing on ethanol could be isolated. Example 3 Functional Testing of Isolated Gene Library Fragments Selected Saccharomyces-clones are cultured twice on complete medium with uracil to test whether the complementation of the chromosomal ICL deletion is plasmid-encoded And the resulting cells were grown as single clones on plates. Of the 16 or 13 randomly selected clones, 7 or 5 no longer grew on minimal medium containing glucose. These clones also did not grow at all on minimal medium with ethanol. Curing of the plasmid correlated with loss of ICL1d complementation. The plasmid was again isolated from one of both clones. It contained approximately 8 kb of insert. New transformation of the Saccharomyces mutant resulted in complementation of all clones found. The 8 kb fragment could be shortened by Sph I to a fully functional 2.9 kb. In a crude extract of a transformant grown on ethanol, isocitrate lyase having a specific activity of 0.3 U / mg of protein could be measured. Furthermore, a Western blot using a polyclonal antibody against Acibia-ICL showed a clear signal. PCR using primers derived from the tryptic peptide of ICL showed a strong signal of the expected magnitude. Proteins were isolated from the two-dimensional electrophoresis gel, digested with trypsin into peptides, and sequenced by Edman degradation (Edmannabbau). Comparison of the peptide sequence with the base data revealed over 70% identity with the isocitrate lyase from Saccharomyces cerevisiae. Primers derived therefrom were used for PCR. Among the complementary gene library fragments of about 8 kb in size, 3.3 kb was sequenced (Sanger et al. Proc. Natl. Acad. Sci. USA 74 (1997) 5463-5467). For the sequences examined, two code ranges were found by comparison of the underlying data. The 1680 base open reading frame (SEQ ID NO: 1) indicates 65% identity to the Saccharomyces cerevisiae ICL1 gene. The ICL gene is located 375 bases upstream of the sequence showing 84% identity to Saccharomyces cerevisiae Ser-tRNA (SEQ ID NO: 1). Example 4: Functionality of Subcloned ICL in E. coli / Yeast / Acibia Shuttle Vector The two fragments obtained by restriction digestion of the isolated gene library fragment and the PCR product (FIG. 5) were analyzed using a Steiner and Steiner It was cloned into the plasmid pAG100 (FIG. 6) constructed by Philippsen (1994, Mol. Gen. Genet 242: 263-271). This fragment is a 2.9 kb SphI-fragment (pAG100ic1.4) and a 2.2 kb Bgl1 / Eco RV-fragment (pAG100ic1.6). Both fragments contained Ser-tRNA. Therefore, additionally, PCR amplification of the putative gene with the Bam HI cleavage site (pAG100icl.8) fused thereto was performed. All three DNAs were cloned at the Bam HI site of plasmid pAG100. The resulting plasmid was used to transform the yeast mutant Saccharomyces cerevisiae ICL1d ura3 (fs). All three constructs made ICL 1d degradation completely complementary, ie, possessed a functional gene. Example 5: Effect of ICL-bearing plasmid on riboflavin formation by Acibia gossypii Transformation of Acibia gossypii using the above-described plasmid (method: Wright and Philippen, 1991, Gene, 109: 99-105) resulted in significant riboflavin formation. An increase was obtained. The culture was carried out in a 500 ml shake flask with two baffles, containing 10 g / l of soybean oil, 10 g / l of yeast extract and 50 ml of a medium consisting of 200 μg / ml of Geneticin. A control strain with a plasmid without insert. Gossypi pAG100 produced 18.7 ± 0.1 mg / l riboflavin in 2 days. Strain A. Gossypi pAG100.4 and A.I. Gossypi pAG100.6 produced 31.2 ± 6.1 mg / l or 31.0 ± 2.0 mg / l of riboflavin (FIG. 7). Significant changes in the specific activity of the isocitrate lyase could not be measured due to strong scattering. Strain A. Gossypi pAG100.8 produced riboflavin 65 ± 5.6 mg / l within 3 days in medium supplemented with 3 g / l glycine. In contrast, the control strain A. Gossypi pAG100 produced only 29.9 ± 1.8 mg / l riboflavin in a direct comparison (FIG. 8). No significant difference could be measured, either in the specific activity of the isocitrate lyase or in the micelle dry weight. Example 6: Purification of isocitrate lyase (ICL) For the identification of ICL inhibitory substances, first the ICL from Acibia gossypii was purified. Isolation and purification of the enzyme performed a 10-fold growth of fungal micelles on vegetable oil. The individual purification steps are summarized in Table 1: according to which a typical crude extract produced from about 25 g of micelles and obtained by cell disruption using a French-press had 220 units of ICL activity. About 78% of it can be dissolved in the supernatant after centrifugation at 40,000 g and reconfirmed. Subsequent ammonium sulfate fractional precipitation gives a three-fold enrichment of the enzyme. After gel filtration on a Sephacryl S-300 column, the TCL is bound to the cation exchanger Mono S-Sepharose and eluted with NaCl. The preparation thus obtained is homogeneous on SDS-polyacrylamide gel electrophoresis and has a specific activity of 18.4 U / mg. Example 7: Identification of an ICL inhibitor Using the purified enzyme, a colorimetric test (Dixon, H. and Kornberg, H. L. (1959), Biochem. J. 72, 3: Assay methods for key enzymes of the glyox ylate cycle), the effect of the substance on the activity can be measured. Table 2 and FIG. 1 summarize and illustrate the effect of the test substances on the enzymes. Substances that may have an inhibitory effect on enzymes as metabolites in fungal cells were examined. Among them, 6-P-gluconate and phosphoenolpyruvate showed a clear inhibitory effect at 50 mM or more at a concentration of 10 mM. However, itaconic acid salts and oxalates, which probably do not occur in fungal metabolism, worked significantly better. A concentration of 1 mM already showed 78% or 95% inhibition. Example 8: Characterization of Mutants Selected in Itaconic Acid Salt Mutations can be induced in genetic material by UV irradiation of isolated fungal spores. At irradiation doses where 10-20% of the spores used survive, mutants are obtained which are resistant to the inhibition of riboflavin production by itaconic acid salts. The mutants so isolated show 25-fold riboflavin formation when grown on soybean oil compared to the starting strain (FIG. 2). The ICL specific activity has been increased to about 15% during the riboflavin forming phase (FIG. 2). Antibodies can be used to show that protein levels are elevated. ICL from the mutant shows similar itaconic acid inhibitory properties as the starting strain. Example 9: Correlation of riboflavin formation and ICL specific activity According to a surprising reference to the causal relationship between ICL and riboflavin formation, when using glucose as a substrate, fungi first produce after glucose consumption. An observation is made to begin. Indeed, ICL, which is presuppressed by glucose, is also measurable in the crude extract and rises to an activity similar to that found on growth on oil (FIG. 3). .

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 645) (C12N 1/15 C12R 1: 645) (C12P 25/00 C12R 1: 645) (81) Designated country EP ( (72) Invention of AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), CA, CN, JP, KR, US E. Hermann Zahm D-52428 Jülich Wendelinstrasse 71 (72) Inventor Klaus-Peter Sterman D-52428 Eurich Wilhelmstrasse 11 (72) Inventor Georg Schmidt D-52457 Alden Hofen Haerstrasse 10 (72) Inventor Teo Bedecker Germany D-52428 Eurich Robert-Koch-Strasse 7 (72) Inventor Harald Zoilberger Germany D-69221 Dossenheim Adalbert-Stifter-Strasse 4

Claims (1)

[Claims] 1. The riboflavin-producing microorganism is cultured in a nutrient medium and subsequently produced. Microbial production of riboflavin by isolating riboflavin For this purpose, use microorganisms with altered endogenous isocitrate lyase (ICL) activity. A process for producing riboflavin microbiologically, characterized in that it is used. 2. Microorganisms have higher ICL activity due to mutations in the endogenous ICL gene 2. The method according to claim 1, comprising an enzyme. 3. Microorganisms have higher ICL gene expression due to increased ICL gene copy number The method of claim 1, wherein 4. An ICL gene comprises a regulatory DNA sequence that enhances gene expression of the ICL gene. The method of claim 1 operably linked. 5. Use of a microorganism having resistance to a substance that inhibits ICL. 2. The method according to 1. 6. 6. The method according to claim 5, wherein the microorganism is resistant to the substances itaconic acid salt or oxalate. The method described. 7. 7. The method according to claim 1, wherein the fungus is used as a microorganism. Method. 8. 8. The method according to claim 7, wherein fungi from the genus Acibia are used. 9. IC coding for the amino acid sequence described in SEQ ID NO: 2 L gene. Ten. A gene construct containing the ICL gene according to claim 9. 11． The ICL gene can be used to increase the expression of one or more regulatory signals and functions. The gene construct according to claim 10, wherein the gene construct is linked together.